JPH0443080B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel physiologically active substance having antitumor activity. In this specification, amino acids and peptides are
Indicated by the abbreviations adopted by the IUPAC-IUB Committee on Biochemical Nomenclature (CBN), for example, the following abbreviations are used. In addition, when an optical isomer is possible for an amino acid, etc., the L-isomer is indicated unless otherwise specified. Ala: Alanine Arg: Arginine Asn: Asparagine Asp: Aspartic acid Gln: Glutamine Glu: Glutamic acid Gly: Glycine His: Histidine Leu: Leucine Lys: Lysine Pro: Proline Ser: Serine Val: Valine Various substances that have reticuloendothelial system activation effects There have been many reports on the existence of substances, for example, substances that are induced by various Gram-positive bacteria and endotoxins and have physiological activities such as antitumor cell ability. for example
Carswell et al.
The serum of the mouse obtained by administering Calmette-Gue'rin (BCG) and intravenously injecting endotoxin 2 weeks later has a cytocidal effect on cultured cells, and that it has a cytocidal effect on cells cultured. We discovered a phenomenon that caused tumors in cancer-induced (BALB/c×C57BL/6) F1 mice to undergo hemorrhagic necrosis.
Named TNF (Tumor Necrosis Fator) [Proc.Nat.Acad.Sci.USA Vol. 72 (No. 9) 3666~
3670 pages (1975)]. Later, Ruff et al [J.
Immunol. vol. 125 (No. 4) pages 1671-1677 (1980)]
and Matthews et al. [Br.J.Cancer 42 vol. 416-422
(1980)] attempted to purify TNF from rabbit serum prepared according to the method of Carswell et al.
The serum was purified approximately 2,000 times and approximately 1,000 times compared to the original serum, respectively. However, in any case, the antitumor and cancer effects of purified products have not been confirmed in animal experiments. JP-A-57-140725 discloses that one or more substances having a reticuloendothelial system activation effect are administered to mammals (mouses, rabbits, guinea pigs, etc.), and then endotoxin derived from Gram-negative bacteria is injected. or by adding endotoxin from Gram-negative bacteria to a tissue culture system containing activated macrophages of mammalian origin.
They disclose that they have isolated and purified a proteinaceous physiologically active substance that has anticancer activity. Furthermore, the molecular weight of the substance was determined to be 39000±5000 by gel filtration method and SDS-polyacrylamide gel electrophoresis, and the isoelectric point was PH3.9.
It is disclosed that it is ±0.3 (isoelectric focusing method). Although the anticancer activity of the above substance is extremely excellent, in order to isolate and purify it, the crude solution must be subjected to a complicated purification process, and the recovery rate of its activity is low. have The present inventors have been conducting intensive research on a purification method for obtaining the above-mentioned substance in good yield from rabbit serum prepared by the method described in this specification, and in the process, By adopting a purification method different from the one described above, a novel protein with excellent antitumor activity, which is different from the above-mentioned substances, was unexpectedly produced in good yield (i.e., the recovery rate through the entire process was approximately 50-70%). As a result of further research and discovery, the present invention was completed. The present invention utilizes a substance having a reticuloendothelial system activation effect.
species or species to rabbits and then induced by injection of endotoxin derived from Gram-negative bacteria or by adding endotoxin derived from Gram-negative bacteria to a tissue culture system containing rabbit-derived macrophages. Ser-Ala-Ser-Arg-Ala-Leu-Ser-
Asp−Lys−Pro−Leu−Ala−His−Val−Val−
Ala−Asn−Pro−Gln−Val−Glu−Gly−Gln−
It has a structure consisting of subunits of a polypeptide having an amino acid sequence structure represented by Leu-Gln-X ₁ -Leu... (wherein X ₁ represents one amino acid residue), and has the following properties: This relates to a protein having the following properties. a) Molecular weight 40000±5000 (gel filtration method) 17500
±2000 (SDS-polyacrylamide electrophoresis method) b) Isoelectric point 5.0±0.3 (isoelectric focusing method) c) Specific activity in evaluation using LM cells described in Experimental Example 3 is 5×10 ⁶ ~1×10 ⁷ units/mg protein d) Meth A The property of causing cancer necrosis in a biological evaluation system using sarcoma tumor-bearing F1 mice The above d) is specifically described in Experimental Example 4.
In biological evaluation using Meth A sarcoma tumor-bearing BALB/c mice, the activity was (+) or higher when 3000 to 5000 units per mouse was intravenously administered. Furthermore, in electrophoresis using a cellulose acetate membrane, the α-globulin region was migrated, and various lectin columns [Con A-Sepharose (manufactured by Pharmacia), RCA-I-gel (manufactured by EY Laboratories), WGA-Sepharose 6MB (Pharmacia), (manufactured by EY Laboratory), UEA-I-gel (manufactured by EY Laboratory), and LPA-gel (manufactured by EY Laboratory). The physical properties of the novel physiologically active substance of the present invention were measured by each method of Experimental Examples 1 to 8 described below. Experimental example 1) Molecular weight measurement method A) Using a column (1.5 x 100 cm) of Cephacryl S-200 (manufactured by Pharmacia, Sweden),
Gel filtration was performed using 0.1M sodium chloride/50mM phosphate buffer (PH7.4). A molecular weight calibration curve was created using standard proteins for molecular weight measurement (manufactured by Pharmacia, ribonuclease A, chymotrypsinogen A, ovalbumin, aldolase), and the molecular weight was measured by activity evaluation using LM cells. The physiologically active substances of the present invention include ovalbumin and chymotrypsinogen A.
It eluted in the middle of , and the molecular weight was 40,000±5,000. B) Method in Segrest et al.
Tris/glycine/SDS (PH8.3) according to Enzymology 28-B, pp. 54-63 (1972)].
10 μg of the sample was applied to an SDS/polyacrylamide gel, and electrophoresis was performed. Create a molecular weight calibration curve using a standard molecular weight kit (manufactured by Pharmacia), evaluate activity in LM cells, and stain (Coomassie Brilliant Blue R-250)
The molecular weight was determined by The physiologically active substance of the present invention showed a single staining band and activity at a position of 17500±2000. 2) Isoelectric focusing method Isoelectric focusing device (SJ
A 5% polyacrylamide flat plate gel (thickness: 1 mm, length: 10 cm, width: 10 cm) containing Pharmalite (manufactured by Pharmacia, PH4 to 6.5) and glycerol was prepared using Polyacrylamide gel (1 mm thick, 10 cm long, 10 cm wide). 0.04M on the anode side,
Pre-phoresis was performed at 700V for 50 minutes using DL-glutamic acid and 0.2M L-histidine on the cathode side. Subsequently, 50μg of sample was applied, and the temperature was set at 700V for 1 hour.
Electrophoresis was performed at 500V for 16 hours. After the electrophoresis is complete, remove the gel.
Cut out 2.5 mm wide pieces, and then soak each gel piece in 0.02 M Tris-HCl buffer (PH) containing 0.15 M sodium chloride.
8.2) Extract with 0.2 ml, and evaluate the activity of each extract using LM cells. The isoelectric point of the physiologically active substance of the present invention was 5.0±0.3. 3) Activity evaluation using LM cells Activity evaluation using LM cells is performed using Ruff
[Edited by Lymphokine Reports Volume 2 E.Pick,
Academic Press 235 pages (1980)] or [J.
Immunol. Vol. 126, p. 235 (1981)], the present inventors have improved the method, and the physiologically active substance of the present invention is derived from L-M cells (American Type Culture Collection, CCL1). 2) to measure the effectiveness of killing. That is, 0.1 ml of the sample was sequentially diluted with the medium and L with a concentration of 10 ⁵ /ml.
- 0.1 ml of the medium suspension of M cells was added to a 96-well tissue culture microplate (Flow Laboratory). Eagle's Minimum Essential Medium containing 1 v/v % fetal bovine serum (its composition is described, for example, in "Tissue Culture" edited by Junnosuke Nakai et al., Asakura Shoten, 1967) was used as the medium. The microplate was incubated at 37°C for 48 hours in air containing 5% carbon dioxide. After culturing, 20μ of glutaraldehyde was added to fix the cells. After fixation, the microplate was washed, dried, and 0.05
0.1 ml of % methylene blue solution was added to stain the surviving cells. After rinsing away excess methylene blue and drying, the remaining methylene blue was extracted with a 0.36M hydrochloric acid solution, and its absorbance at 665 nm was measured using Titertech Multiscan (Flow Laboratory). This absorbance is proportional to the number of surviving cells. The amount of biological activity required to kill 50% of LM cells is defined as 1 unit/ml, and the dilution rate of the sample corresponding to 50% of the absorbance of the control without the sample added is shown in the graph or calculation. The reciprocal of the dilution rate was determined as the physiological activity amount (expressed in units/ml) of the sample. On the other hand, the protein amount was determined by the method of Branford et al. [Anal.
Biochem., Vol. 72, pp. 248-254 (1976)], from a dye binding method using Coomassie Brilliant Blue G250. The specific activity of the physiologically active substance of the present invention is 5×
It was 10 ⁶ to 1×10 ⁷ units/mg protein. 4) Activity evaluation using Meth A sarcoma ^tumor -bearing mice.
Meth A sarcoma cells were transplanted, and after 7 days, the transplanted tumor size was 7 to 8 mm in diameter, and mice with tumors with good blood circulation without hemorrhagic necrosis were selected and diluted with physiological saline from the tail vein. 0.2
ml of the sample was injected, and 24 hours later, the necrosis reaction was judged according to the following criteria. (-): No change (+): Faint hemorrhagic necrosis (): Moderate hemorrhagic necrosis (necrosis extending from the center of the transplanted cancer surface to more than 50%) (): Significant hemorrhagic necrosis (transplanted cancer The central part of the sample was severely necrotic, with only a small amount of surrounding cancer tissue remaining.20 days after sample administration, whether the cancer had completely regressed was observed and the complete cure rate was determined. The activity of this physiologically active substance measured by the above method is shown in the table below.

[Table] Salt water)

Claims (1)

[Claims] 1. By administering to a rabbit one or more substances having a reticuloendothelial system activation effect, and then injecting endotoxin derived from Gram-negative bacteria,
or Ser-Ala-Ser-Arg-Ala-Leu-Ser-Asp from the amino terminus induced by adding endotoxin from Gram-negative bacteria to a tissue culture system containing rabbit-derived macrophages.
−Lys−Pro−Leu−Ala−His−Val−Val−Ala
−Asn−Pro−Gln−Val−Glu−Gly−Gln−Leu
-Gln-X ₁ -Leu... (However, in the formula, X ₁ represents one amino acid residue.) Having a structure consisting of subunits of a polypeptide having the amino acid sequence structure
and a protein having the following properties. a) Molecular weight 40000±5000 (gel filtration method) 17500±2000 (SDS-polyacrylamide electrophoresis method) b) Isoelectric point 5.0±0.3 (isoelectric focusing method) c) Use L-M cells as defined in the text Specific activity in evaluation is 5 x 10 ⁶ to 1 x 10 ⁷ units/mg protein. d) Property of causing cancer necrosis in a biological evaluation system using Meth A sarcoma tumor-bearing F1 mice